mTOR- and LARP1-dependent regulation of TOP mRNA poly(A) tail and ribosome loading [polysome fractionation]. mTOR- and LARP1-dependent regulation of TOP mRNA poly(A) tail and ribosome loading [polysome fractionation]

Translation of TOP mRNAs encoding protein synthesis machinery is strictly regulated by an amino acid sensing mTOR pathway. However, its regulatory mechanism remains elusive. Here, we demonstrate that TOP mRNA translation positively correlates with its poly(A) tail length under mTOR active/amino acid-rich condition, suggesting that TOP mRNAs are post-transcriptionally controlled by poly(A) tail length regulation. Consistent with this, tail length of TOP mRNAs dynamically fluctuates in response to amino acid availability. Poly(A) tail shortens under mTOR active/ amino acid-rich condition, whereas the long-tailed TOP mRNAs accumulate under mTOR inactive/amino acid-starved (AAS) condition. An RNA-binding protein LARP1 that specifically binds to TOP mRNAs is indispensable for the process. We also show that LARP1 interacts with non-canonical poly(A) polymerases, PAPD4, PAPD5 and PAPD7 and induces post-transcriptional polyadenylation of the target when tethered to the mRNA. Our findings illustrate that LARP1 contributes to the selective accumulation of TOP mRNAs with long poly(A) tail under AAS, resulting in accelerated ribosomal loading onto TOP mRNAs for the resumption of translation after AAS. Overall design: 5’ capped polyA RNAs purified from free, monosomal, light-polysomal and heavy-polysomal fractions were analyzed by direct RNA-sequencing using the Oxford Nanopore’s MinION.

编码蛋白质合成装置的5'端寡嘧啶mRNA（TOP mRNA）的翻译过程，严格受氨基酸感应型雷帕霉素靶蛋白（mTOR）通路调控，但其具体调控机制至今尚未明确。本研究证实，在mTOR激活、氨基酸充足的条件下，TOP mRNA的翻译效率与其poly(A)尾长度呈正相关，提示TOP mRNA的表达受转录后poly(A)尾长度调控。与此一致，TOP mRNA的poly(A)尾长度会随氨基酸可获得性发生动态变化：在mTOR激活、氨基酸充足时，TOP mRNA的poly(A)尾会缩短；而在mTOR失活、氨基酸饥饿（AAS）条件下，携带长poly(A)尾的TOP mRNA会发生积累。特异性结合TOP mRNA的RNA结合蛋白LARP1，是该调控过程不可或缺的关键因子。本研究同时发现，LARP1可与非经典poly(A)聚合酶PAPD4、PAPD5及PAPD7相互结合，当LARP1被锚定至靶mRNA时，可介导靶mRNA的转录后多聚腺苷酸化。本研究结果表明，LARP1可在氨基酸饥饿条件下介导携带长poly(A)尾的TOP mRNA选择性积累，进而提升核糖体在TOP mRNA上的装载效率，助力氨基酸饥饿解除后翻译过程的恢复。实验整体设计：从游离核糖体、单核糖体、轻多核糖体及重多核糖体组分中纯化得到的5'帽化polyA RNA，采用牛津纳米孔MinION测序仪开展直接RNA测序分析。